The ability of cells, tissues, an organ system and an entire organism to rapidly respond and adapt to exogenous stimuli is a requirement for the maintenance of life. Exposure of a single cell to such stimuli can manifest itself in a variety of ways, including a flux of essential intracellular ions (i.e. Na+, K+, Ca++, Cl-, H+), as well as changing oxygen and glucose levels. These changes can trigger additional signaling cascades, ultimately resulting in the recruitment of the appropriate cellular machinery for a response to the stimuli.
Of course, some stimuli are pathogenic to cells. Such stimuli cause a combination of linked and cascading biochemical events leading up to disease and/or cell death. For example, exposure to bacteria, viruses, toxins and toxicants may result in a myriad of intra/extracellular responses, depending on the pathogen or pathogenic agent in question and the route of exposure. The determination and understanding of which of the "downstream" biochemical signals elicited are indicators of physical, chemical or mechanical injury are fundamental to the development of countermeasures and therapy.
Classical biochemical investigations of the toxicologic effects of chemicals on organs and tissues were typically performed on homogenates. This approach reduced complex arrays of cells to a uniform blend. While providing important new information on fundamental mechanisms of toxicology/pharmacology, these studies are limited in their ability to discriminate between cells which are passively or actively involved.
More recent molecular and imaging techniques have improved cellular resolution. However, these newer imaging techniques frequently provide only static "snapshots" of dynamic cellular processes. Other approaches, while more dynamic, suffer from the fact that the approach alters the cells under study. For example, commercially available fluorescent probes used in the detection of calcium fluxes, chemically bind the moiety in question and potentially alter its homeostasis in situ.
Clearly, the most extensive work done intracellularly focused on the direct injection of dyes into the cell. While this method has provided researchers with a simple technique to study cellular processes, it has also proven problematic. For instance, the dye may itself be toxic, or otherwise interfere with the cell chemistry. Another problem is that there is no way to position the dye once it is introduced into the cell. Often, the dye is selectively trapped in some organelles, rather than dispersed evenly throughout the cell.
An additional, critical limitation with the dye injection approach is that the technology is currently limited in selectivity to a small number of analytes. For instance, while there are good dyes for calcium ion detection, there are none for potassium, sodium or chloride.
Fiber optic probes, or optodes, with a polymer sensing element, solve the above problems of dye injection. See W. Tan et al., "Submicrometer Intracellular Chemical Optical Fiber Sensors," Science 258:778 (1992). These micro-fiberoptic sensors (100-1000 nm) are based on optical grade silica fibers pulled to submicron size. The pulled fiber tips are much less fragile than those of the electrochemical microsensors, which are made from pulled micropipettes. Attached to the tip is a dye-polymer matrix, which is very durable and smooth and runs tightly bound to the tip, even during penetration of biological tissues. The matrix on the end of the fiber often includes several components, such as a chromoionophore, an ionophore, and appropriate ionic additives, all trapped inside a polymer layer, so that no chemicals are free to diffuse throughout the cell. The effects of toxicity of the dyes are thus minimized. Also, the probe can be carefully positioned in the cell, allowing any specific area to be imaged or monitored.
Nonetheless, the fiber optic probes have the significant drawback of being unable to easily monitor more than one location in the cell. For monitoring more than one location, multiple probes are needed. Due to size constraints, it can prove difficult to position several fibers inside a single cell. Moreover, even the insertion of single fiber sensor can easily damage a cell or short out the cross membrane electrical potential and having several fibers compounds this problem.
Thus, improved methods for studying cells and intracellular analytes are needed. Such improved methods should be amenable to monitoring the cell at more than one location and should have minimal toxicity.